Intramolecular cross-linking of proteins by formation of lysinoalanine or lanthionine. Modification of disulfides in ovomucoids.

Abstract

The disulfide bonds of ovomucoids were cleaved and new sulfur‐containing cross‐links were introduced by two separate chemical modification methods: (1) alkali treatment, and (2) cyanolysis. Alkali and cyanolytic treatments were used to cleave disulfide bonds and to introduce new synthetic nonreducible cross‐links consisting of residues of lysinoalanine (N‐(dl‐2‐amino‐2‐carboxyethyl)‐l‐lysine) and lanthionine (bis(2‐amino‐2‐carboxyethyl)sulfide). The two ovomucoids studied were turkey and penguin ovomucoids, which are “double‐headed” inhibitors of proteolytic enzymes with one inhibitory site for bovine trypsin and another for bovine α‐chymotrypsin or subtilisin. Trypsin does not compete with either α‐chymotrypsin or subtilisin, but the latter compete with one another, apparently for the same inhibitory site. The stability of inhibitory activities as a function of disulfide bond scission and the formation of new nondisulfide cross‐links was studied. In both methods of disulfide modification of turkey ovomucoid, inhibitory activity against trypsin was more stable with respect to the extent of modification than was inhibitory activity against chymotrypsin or subtilisin. With penguin ovomucoid, inhibitory activity against subtilisin was always more stable than activity against trypsin or chymotrypsin with both methods. With both ovomucoids it was thus possible to produce single‐headed inhibitors from the double‐headed inhibitors. The formation of the nonreducible cross‐links of lanthionine from cystines, and of lysinoalanines from cystines and lysines, with retention of a biochemical activity, suggests that such a procedure may have at least limited use as a cross‐linking method.